Identification of Electrode Respiring, Hydrocarbonoclastic ... · by measuring the reduction of...

ORIGINAL RESEARCHpublished: 09 December 2016

doi: 10.3389/fmicb.2016.01965

Frontiers in Microbiology | www.frontiersin.org 1 December 2016 | Volume 7 | Article 1965

Edited by:

Yong Xiao,

Institute of Urban Environment

(Chinese Academy of Sciences),

China

Reviewed by:

Yingying Wang,

Nankai University, China

Shaohua Chen,

Agency for Science, Technology and

Research, Singapore

*Correspondence:

Krishnaveni Venkidusamy

krishnaveni.venkidusamy@

mymail.unisa.edu.au

Specialty section:

This article was submitted to

Microbiotechnology, Ecotoxicology

and Bioremediation,

a section of the journal

Frontiers in Microbiology

Received: 09 September 2016

Accepted: 24 November 2016

Published: 09 December 2016

Citation:

Venkidusamy K and Megharaj M

(2016) Identification of Electrode

Respiring, Hydrocarbonoclastic

Bacterial Strain Stenotrophomonas

maltophilia MK2 Highlights the

Untapped Potential for Environmental

Bioremediation.

Front. Microbiol. 7:1965.

doi: 10.3389/fmicb.2016.01965

Identification of Electrode Respiring,Hydrocarbonoclastic Bacterial StrainStenotrophomonas maltophilia MK2Highlights the Untapped Potential forEnvironmental BioremediationKrishnaveni Venkidusamy 1, 2* and Mallavarapu Megharaj 1, 2, 3

1Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, SA, Australia,2Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Mawson Lakes, SA,

Australia, 3Global Centre for Environmental Remediation, The University of Newcastle, Callaghan, NSW, Australia

Electrode respiring bacteria (ERB) possess a great potential for many biotechnological

applications such as microbial electrochemical remediation systems (MERS) because of

their exoelectrogenic capabilities to degrade xenobiotic pollutants. Very few ERB have

been isolated from MERS, those exhibited a bioremediation potential toward organic

contaminants. Here we report once such bacterial strain, Stenotrophomonas maltophilia

MK2, a facultative anaerobic bacterium isolated from a hydrocarbon fed MERS, showed

a potent hydrocarbonoclastic behavior under aerobic and anaerobic environments.

Distinct properties of the strain MK2 were anaerobic fermentation of the amino acids,

electrode respiration, anaerobic nitrate reduction and the ability to metabolize n-alkane

components (C8–C36) of petroleum hydrocarbons (PH) including the biomarkers, pristine

and phytane. The characteristic of diazoic dye decolorization was used as a criterion for

pre-screening the possible electrochemically active microbial candidates. Bioelectricity

generation with concomitant dye decolorization in MERS showed that the strain is

electrochemically active. In acetate fed microbial fuel cells (MFCs), maximum current

density of 273 ± 8 mA/m2 (1000) was produced (power density 113 ± 7 mW/m2) by

strain MK2 with a coulombic efficiency of 34.8%. Further, the presence of possible alkane

hydroxylase genes (alkB and rubA) in the strain MK2 indicated that the genes involved

in hydrocarbon degradation are of diverse origin. Such observations demonstrated

the potential of facultative hydrocarbon degradation in contaminated environments.

Identification of such a novel petrochemical hydrocarbon degrading ERB is likely to offer

a new route to the sustainable bioremedial process of source zone contamination with

simultaneous energy generation through MERS.

Keywords: electrode respiring bacteria, microbial electrochemical remediation systems, Stenotrophomonas

maltophilia MK2, facultative hydrocarbon degradation, dye decolorization, catabolic genes (alkB, rubA)

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Venkidusamy and Megharaj S. maltophilia: Implications in Environmental Bioremediation

INTRODUCTION

Due to their toxicity and ubiquitous nature, petroleumhydrocarbons (PH) are of serious concern to the environmentaland public health. Of these PH contaminants, diesel rangeorganics (DRO) is constitute one of the most prevalent organicpollutants that are biodegradable in various environments.Medium chain hydrocarbons from octane to the long chainhydrocarbon dotriacontane are the constituents of DRO. Theyare usually assumed to be the fractional middle distillate ofcrude oil and are known to be highly noxious, hazardous,and carcinogenic (Chilcott, 2011). Increasing anthropogenicactivities of these compounds leading to spillages, and leakagesfrom underground storage tanks constitute the two dominantsources of penetration of DRO compounds from surface soilsto subsurface. As an ultimate result, DRO became the mostencountered environmental pollutants in groundwater and soils(Gallego et al., 2001). Consequently, horizons of subsoil, aquiferand groundwater systems are prone to long-term contaminationof these hydrophobic contaminants. Microbial clean-up of theseDRO compounds is claimed to be an efficient, economical,and versatile alternative to the established physicochemicaltreatments that are prone to cause recontamination by secondarycontaminants (Hong et al., 2005; Megharaj et al., 2011). Thebiodegradation of these compounds at the surface has been welldocumented for a century whereas subsurface biodegradationawaits further research on deeper insights into the metabolicactivities involved and the extent and rate of hydrocarbondegradation (Röling et al., 2003). Subsurface hydrocarboncontaminated reservoirs are primarily dominated by obligate andfacultative anaerobic microbial communities. These microbialcommunities can adjust their metabolism to take account of theavailability of final electron acceptors and can havemore complexenzymatic systems involved in the degradation of contaminants.However, the rate ofmicrobial utilization of these PH compoundsis very slow especially under anaerobic environments where theavailability of relevant electron acceptors is limited (Morris et al.,2009).

Recent research on removal of such recalcitrant contaminantsusing advanced microbial electrochemical systems is gainingnew interest in its practical applications involved in subsurfacehydrocarbon bioremediation. These microbial electrochemicalremediation systems (MERS) transform the chemical energyavailable in organic pollutants into electrical energy bycapitalizing on the biocatalytic potential of a peculiar groupof microbes called “electric communities” (Logan, 2008; Morriset al., 2009). These electric microbial communities have receivedmuch attention in the field of electromicrobiology becauseof their exoelectrogenic capabilities to degrade substrates thatrange from easily degradable natural organic compounds toxenobiotic compounds such as PH contaminants (Venkidusamyand Megharaj, 2016; Venkidusamy et al., 2016; Zhou et al.,2016). Many studies have shown the predominance of manystrains and species of Geobacter in microbial fuel cells(MFCs) fed with different types of substrates. However, themicrobial community composition is divergent in MERS(Morris et al., 2009; Venkidusamy et al., 2016), and the

physiology of such populations remains to be explored indetail. The identification of such bacterial population withdual functions of electrode respiration and petrochemicaldegradation highlights the biotechnological potential involvedin sustainable remediation of PH contaminated sites andMERS. We have therefore attempted to (i) find representativemicrobial candidates with such abilities of hydrocarbonoclasticelectrode respiration through the anode enrichment ofMERS, (ii)demonstrate the bioremediation potential of isolated bacteria tocompletely mineralize DRO compounds in anoxic environmentsand (iii) also investigate the presence of catabolic genesresponsible for hydrocarbon degradation in these bacteria.

MATERIALS AND METHODS

Source of ChemicalsRefined fossil fuels such as DRO and other PH productsused throughout the study were obtained from local BP outlet(Australia). Aliphatic hydrocarbon standards, solvents such ashexane and methylene chloride, redox indicators such as 2–6,dichlorophenol indophenol (DCPIP), and tetrazolium violet (2,5-diphenyl-3-[α-naphthyl] tetrazolium chloride) and diazo dyeswere purchased from Sigma Aldrich Trading Co. Ltd (Australia).All the solvents used were of HPLC grade.

Bacterial Strain, Media, and CultureConditionsThe bacterial strain MK2 was isolated from the anodic biofilmof a MERS fed with hydrocarbons operated in a fed-batchmode over a period of 12 months. Hydrocarbons contaminatedgroundwater (RAAF Base, Williamstown, NSW, Australia) andactivated sludge (WTP, South Australia) served as inoculum forthese PH fed MERS. Bacterial cells from the anodic biofilm wereextracted into a sterile phosphate buffer and shaken vigorouslyto separate cells from the electrode. Aliquots of the extractedcell suspensions were serially diluted and plated onto mineralsalt medium (MSM) agar (Grishchenkov et al., 2000) containing1% DRO compounds and incubated for 3 weeks. Single colonieswere selected and transferred to Luria Bertani (LB) agar plates.Unless otherwise stated all incubations were performed at roomtemperature. Media used throughout the study were BushnellHass (Hanson et al., 1993), mineral salts medium (Grishchenkovet al., 2000) and Luria-Bertani medium (Sambrook et al., 1989).Nitrate served as the terminal electron acceptor in anaerobicbiodegradation experiments. A chemically defined mediumsupplemented withWolfe’s trace elements and vitamins was usedin the microbial electrochemical studies as previously described(Oh et al., 2004). One liter of growthmedium contains (g l−1) KCl0.13, Na2HPO4 4.09, NaH2PO4 2.544, NH4Cl 0.31. The pH of themediumwas adjusted to 7± 0.2 and further fortified withWolfe’strace elements and vitamins. The purified strain was stored inglycerol: Bushnell Hass broth and glycerol: Luria-Bertani broth(1:20) at −80◦C. Biolog-GN2 (Biolog., USA) plates were used todetermine the utilization of various carbon sources according tothe manufacturer’s instructions.


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Bacterial 16S rRNA Gene SequencingGenomic DNA of strain MK2 was extracted from aerobicallygrown cells using the UltraClean microbial DNA isolationkit (MO BIO, CA, USA) following the manufacturer’sinstructions. The polymerase chain reaction (PCR)mediated amplification of 16S rRNA gene fragments wasperformed using the combination of universal primers,E8F (5′-AGAGTTTGATCCTGGCTCAG3′) and 1541R(5′AAGGAGGTGATCCANCCRCA 3′) (Weisburg et al.,1991). The PCR products were purified using the UltraCleanPCR clean-up kit (Mo Bio, Carlsbad, CA, USA) following themanufacturer’s instructions and sequenced in both directionsusing an automated sequencer, ABI3130 Sequencer (AppliedBiosystems, USA) at the Southern Pathology Sequencing Facility,Flinders Medical Centre (South Australia). 16S rRNA sequenceswere analyzed using the BLAST programme against the NCBIdatabases. The highest hit obtained through blastn match forthe strain MK2 was used for ClustalW multiple alignment andgenerating a phylogenetic relationship. The neighbor joiningtree was constructed using the molecular evolutionary geneticanalysis package version 5.0 based on 1000 bootstrap values(Tamura et al., 2011). The 16S rRNA sequence of strain MK2 wasdeposited in GenBank under accession number JQ316533.

Assessment of Biodegradation Potentialand Electrochemical ActivityThe hydrocarbonoclastic potential of strain MK2 was evaluatedby measuring the reduction of metabolic indicators such asdichlorophenol indophenol and tetrazolium salts (Pirôllo et al.,2008). Experiments were also conducted to pre-screen thepossible candidate electroactive bacterial strains by in vivobiodecolourization assay using diazo dyes as stated earlier (Houet al., 2009). Experiments were carried out in both aerobic andanaerobic environments using 20 ml of nutrient broth withdifferent concentrations (50, 100, 150mg l−1) of an azo dye,Reactive Black5 (RB5). The dye degradation was monitoredby observing the decrease in absorbance of suspension at 595nm under a UV-visible spectroscopy system (Agilent model8458). All decolorization studies were conducted in triplicate foreach experiment, and the activity was expressed as percentagedegradation as follows:

Percentage of dye decolourization =Ai − At

Ai× 100

where Ai = initial absorbance and At = observed absorbance atdesignated intervals.

Hydrocarbon Biodegradation ExperimentsTo obtain 1 OD culture, overnight grown bacterial cells werecentrifuged for 20 min at 4500 rpm. The cell pellet was washedthree times and re-suspended in MSM until the OD600 wasequivalent to 1.00. One percent of the 1 OD culture of strainMK2 was transferred to 100 ml of MSM with a concentrationof 8000mg l−1 of DRO and incubated at 25 ◦C for timecourse experiments with shaking at 150 rpm. The cell growthwas determined by the comparison of optical density against

the control at designated time intervals. Hydrocarbonoclasticpotential was also monitored under anaerobic nitrate reducingenvironments. The inoculum size was 1% of the anaerobicallygrown bacterial cells with nitrate (10 mM) and 8000mg l−1 ofDRO as an electron acceptor and donor, respectively from ananoxic sterile stock solution. All cell cultures were maintainedin triplicate for each experiment. All procedures for anoxicgrowth experiments, from medium preparation to manipulatingthe strain were performed using standard anoxic conditions. Allculturing was done in sealed serum vials with nitrogen/carbondioxide (80:20, v/v) in the headspace. The sealed vials wereincubated at 25◦C for time course experiments with shaking at150 rpm. An uninoculated control was prepared for each set ofbiodegradation experiments. The samples from the time courseexperiments of aerobic and anaerobic incubations were extractedthree times with 1:1 solvent mixture of acetone-methylenechloride, dewatered and concentrated by an evaporator. Theevaporated hydrocarbons were taken as residual hydrocarbonsand dissolved in n-hexane, filtered through 0.25 µm membranefilters and analyzed by gas chromatography.

Fuel Cell ExperimentsMFC Construction and Operational ConditionsSingle chamber MFC systems were constructed from laboratorybottles (320 ml capacity, Schott) as previously described(Logan et al., 2007) with a modification to increase electrodearea. The anode electrodes composed of carbon fiber brusheswith wire titanium cores that had an initial surface area of6.99m2 g−1. These fiber electrodes were cleaned by soakingovernight in acetone followed by pre-treatment with sulfuric acid(concentrated, 100 ml l−1) and heat treatment to improve thegeometric surface area of the electrodes as described by Fenget al. (2010). The cathode was fabricated using flexible carboncloth coated with a hydrophobic PTFE layer (Cheng et al., 2006)with additional diffusional layers on the air breathing side to cutdown fouling rate and evaporation of hydrocarbons. In contrast,the hydrophilic side was coated using a mixture of nafionperfluorinated ion exchange ionomer binder solution, carbon,and platinum catalyst (0.5 g of 10% loading). The electrodes wereconnected using copper wire with all exposed metal surfacessealed with a non-conductive epoxy resin (Jay Car, Australia). Allthe reactors were sterilized before use. Strain MK2 was used formicrobial electrochemical experiments with acetate (1 g l−1) asthe electron donor in 50 mM PBS buffer. The anodic chamberwas flushed for 30 min with nitrogen gas before the operation.The anolyte was agitated using a magnetic stirrer operating at100 rpm. Open circuit MFC studies were also carried out andthen switched to the closed circuit with a selected external load(R-1000 unless stated otherwise). Reactive Black 5 was used assole source of energy in dye degradation experiments using thestrain MK2 at a concentration of 50mg l−1 in MFC studies. LBmedium was used in biodecolorization studies with an externalload of 1000 . MFCs were operated in a fed-batch mode untilthe voltage fell to a low level (≤10 mV), and then the anolytesolution was replaced under anaerobic chamber (10% hydrogen,10% carbon dioxide and 80% nitrogen) (Don Whitley Scientific,




MG500, Australia) conditions. All the reactors were maintainedat room temperature in triplicates.

Cloning and Phylogenetic Analysis ofPossible Catabolic Genes for HydrocarbonDegradationGenes encoding alkane hydroxylase enzyme complex includingalk and rub genes were amplified by a polymerase chain reaction(PCR) method using oligonucleotides listed in Table S1. ThePCR mix of 50 µl contained the following: 10 µl of Gotaq 5Xbuffer, 2.0 µl of MgCl2 (25 mM), 1 µl of dNTP mix (10 mM),2 µl of each primer (100 mM), 10–15 ng of purified DNA and2.5 U taq DNA polymerase (Promega, Australia). Cycling wasperformed with an initial denaturation for 5 min, followed by35 cycles of 60 s at 94◦C, 30 s of annealing at 40–60◦C, 60 s ofextension at 72◦C and a final extension at 72◦C for 10 min,using a Bio-Rad thermal cycler. The primers were designed basedon the available draft genomes of S. maltophilia using Primer—BLAST tool from NCBI and assessed by Oligo 6 software. Theamplification products were purified using the UltraClean PCRclean-up kit (Mo Bio, CA) and ligated into the PGEM-T-Easyvector. After transformation into E. coliDH5α individual plasmidinserts were sequenced. In silico analysis was done by usingthe blast programs to search the GenBank and NCBI databases(http://www.ncbi.nlm.nih.gov).

Analytical Methods and CalculationsCell voltage was monitored using a DMM (Keithly Model 2701,USA) linked to a multi-channel scanner (Module 7700, KeithlyInstruments, USA). Data were recorded digitally on an Intelcomputer via IEEE 488 input system and Keithly cable. Tomeasure the current under closed circuit conditions, the externalresistance was connected (R-1000 unless stated otherwise).Polarization curves were obtained using various external loadsranging from 10 to open circuit. Current was calculated byusing I = V/R. The power density was calculated as follows;where V was the cell voltage, I was electrical current and Adenoted the electrode surface area. Power density and currentdensity were normalized to the projected surface area of a cathode(Logan, 2008).

P =V · I

A(1)

Coulombic efficiency (CE) was calculated at the end of thecycle from COD removal as follows (Logan, 2008),

CE (%) =M

∫ t0 I · dt

Fbq1COD× 100 (2)

where, M is the molecular weight of the substrate, F = Faraday’sconstant, b = number of electron exchanged/1 M of oxygen, Vn= volume of liquid in the anode chamber, 1COD= difference inthe COD of initial and end batch samples from MFCs. Graphitefiber surface area was also measured using a Brunauer–Emmett–Teller (BET) isotherm (Mi micromeritics, Gemini V, Particle andSurface Science Pty Ltd.) DRO degradation experiments wereconducted using data from triplicate analyses. The DRO was

extracted in acetone-methylene chloride (1:1)mixture, dewateredand concentrated by an evaporator, and then analyzed with GC-FID (Flame Ionization Detector) using an HP-5 capillary column(15m length, 0.32 mm thickness, 0.1 µm internal diameter)(USEPA, 1996). The estimated recovery was more than 70%.TheGC programme was set up according to USEPA (USEPA, 1996).The carrier gas was helium. The operational temperature rangedfrom 50 to 300◦C with a programmed temperature gradient of25◦C/min. The resulting chromatograms were analyzed usingAgilent software (GC-FID Agilent model 6890) to identify thepetroleum degradation products (Venkidusamy et al., 2016).

RESULTS

Strain Isolation and PhysiologyFrom the anodes of enriched PH fed MERS, a pure cultureof facultative, hydrocarbonoclastic bacterial strain MK2 wasisolated by serial dilution and plating techniques. Cells of strainMK2 contains double membrane bilayers, produces polar flagellain tufts or as single (Figure 1A) and grow as bacillus shaped(Figure 1B). Cell growth on nutrient agar medium produceslarge gleaming colonies which are pale yellow in color. Thebacterial strain grew at temperatures ranging from 25 to 37◦Cat a neutral pH (optimum temperature 30◦C), while no growthwas detected above 40◦C. The strain was negative for oxidase andcatalase is present. The bacterial strain was shown to be capableof anaerobic growth through amino acid fermentation andanaerobic nitrate reduction through quantitative biochemicalanalysis. However, it was unable to metabolize sugars such asglucose and lactose through the anaerobic fermentation process.Cell growth was accompanied by the strong ammonia odorwith pale green discoloration in old LB plates. The strain MK2displayed a limited nutritional spectrum as highlighted by itsgenus name (Table S2). The strain was unable to utilize arabinose,adonitol, fructose, xylose, rhamnose, gluconate, etc., Salientproperties of the strainMK2were direct electrode respiration andthe ability to degrade n-alkane components of PH in both aerobicand anaerobic environments.

Phylogenetic Analysis and TaxonomyAn almost complete 16S rRNA gene sequence (1448 bp)was obtained for strain MK2 and analyzed phylogeneticallyusing ClustalW alignment. Using this multiple alignment, theneighborhood phylogenetic tree was constructed (Figure 2). Thetaxonomic position shows that the strain MK2 was a member ofthe Stenotrophomonas subgroup in the class of γ-proteobacteria.From a BLAST analysis, the highest level of sequence similarity(98%) matched with Stenotrophomonas maltophilia strain ATCC13637.

Redox Indicator Assays for theAssessment of HydrocarbonoclasticPotentialThe hydrocarbonoclastic potential of strain MK2 wasassessed through a preliminary investigation of hydrocarbonconsumption, a concomitant increase in biomass and reductionof redox electron acceptors such as DCPIP and tetrazoliumindicators. The strain MK2 discolored the redox indicator from


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FIGURE 1 | Transmission electron micrographs of S. maltophilia MK2. Bar scale, 500 nm. (A) Cells with flagella. (B) Bacillus shaped cells of strain MK2.

FIGURE 2 | Phylogenetic tree based on 16S rRNA sequences showing the positions of the strain MK2 and representatives of other Stenotrophomonas

spp. The tree was constructed from 1448 aligned bases. Scale bar represents 0.005 substitution per nucleotide position.

the blue to violet during the first 24 h and complete discolorationwas observed by the end of 120 h when DRO was the sole carbonand energy source. Also, the formation of a red precipitateformazan from the tetrazolium was observed while the abioticcontrols remained unchanged. It is evident from the abovescreening assays that the strain MK2 can utilize diesel derivedhydrocarbons.

Screening Assays for the Assessment ofElectrochemical ActivityTo pre-screen the electrochemical activity of the strain MK2,aerobic and anaerobic cultures were grown in nutrient broth

supplemented with 50mg l−1 of RB5. This concentration wasfound to be supportive for a higher growth rate and rapiddecolorization among the various concentration of RB5 tested.The complete disappearance of the characteristic absorptionpeak at the region of λmax (597 nm) and simultaneousdecolorization were observed in aerobic and anaerobicallyincubated samples (Figure 3). Figure 3A shows dynamic changesof the absorption spectra observed during the decolorizationprocess under anaerobic conditions. RB5 azoic dye was almostcompletely decolorized (96.23%) in 48 h by S. maltophilia MK2under anaerobic environments while it took 72 h for nearlycomplete decolorization (97.99%) under aerobic conditions




(Figure 3B). The blue pigmented dead cell pellet from theheat-killed cells in the control showed a passive adsorptionof dye, whereas colorless cell pellets obtained from the livingcultures demonstrated that reduction of the RB5 indicator hadoccurred.

Energy Generation by S. maltophilia MK2in Microbial Electrochemical CellsCurrent Generation in Acetate Fed MFCsCurrent was generated in all the MFCs inoculated with S.maltophilia MK2 within a few hours using acetate as an energysource. After 3 days, voltage started to follow a constant patternand then stabilized. The fuel cell electrodes were connectedthrough a resistor (R = 1000 ) once it reached the plateauvoltage generation stage. The maximum output range of voltageand current density were 414 ± 7 mV, 273 ± 8 mA/m2 (R =1000 ) after four cycles of operation. After five refilling batcheswith a fresh substrate, the maximum current output of eachbatch became stable (270± 5 mA/m2). Few representative cycles(average current density from triplicates) of current densityare shown in Figure 4A. The maximum CE was 34.8% whichcorresponded to the maximum current density of 272.96 mA/m2.

Current Generation and Simultaneous Dye

Decolorization in Dye Fed Microbial Electrochemical

CellsThe current was rapidly generated in azo dye fed MFCsinoculated with S. maltophilia MK2 cells within few hours ofusing azoic dye as an energy source at 1000 . The maximumoutput range of voltage and current density were 145 ± 6mV, 94 ± 6 mA/m2. Constant and repeatable power cycleswere obtained during five changes of the contents of the anodechamber. Using RB5 concentration of 100mg l−1 in MFC, 59.3± 1.25% was removed during the first 12 h of operation. After24 h, almost 97.2 ± 1.64% of RB5 was decolorized and itwas below detection limits at the end of the batch operationwhen the voltage of the batch reached >10 mV as shown inFigure 4B.

Hydrocarbonoclastic Potential ofS. maltophilia MK2Aerobic Biodegradation of DROTo evaluate the hydrocarbon degradation potential of thestrain MK2, experiments were performed under two differentenvironments viz., aerobic and anaerobic. The rate and extent

FIGURE 3 | (A) Time overlaid absorbance spectra of RB5 biodecolourization by the strain MK2. (B) Biodecolourization of diazoic dye RB5 by the strain S. maltophilia

MK2 under aerobic and anaerobic environments.

FIGURE 4 | (A) Few representative cycles of current density generated by S. maltophila MK2 in acetate fed microbial fuel cells. (B) Current generation and

simultaneous dye decolorization in dye fed MERS using S. maltophila MK2.




of biodegradation were interpreted from GC chromatograms ofthe residual hydrocarbons. The aerobic incubation experimentsindicated that the biodegradation of hydrocarbons by strainMK2was more efficient than anaerobic incubations. Figure 5A showsthe possible cell growth and its associated substrate degradationby strain MK2. For a substrate concentration of 8000mg l−1,cells started growing within 24 h with a rapid decrease in DROconcentration of about 53%. After 84 h, the strain reachedas second peak of growth while the DRO degradation was88%. The temporal removal of DRO reached >90% after 100h of incubation. In general, the rate of degradation increasedconsistently with increasing cell biomass during the early stageof the exponential phase and then, it reached a plateau atstationary and death phase of cell growth. Abiotic loss of DROwas measured under each stage was less than 5%. The GCprofile of the residual n-alkanes of DRO after the incubation wascompared with that of the original as shown in Figure S1. At theend of the incubation period (150 h), the n-alkane members ofC8 to C36 were almost completely metabolized in the samplesinoculated with the strain MK2.

Anaerobic Biodegradation of DROThe hydrocarbonoclastic activity of the strainMK2was examinedunder anaerobic conditions with DRO as the sole sourceof carbon and nitrate as the final electron acceptor. Theresults indicated that the biodegradation of DRO (8000mgl−1) in anaerobic environments is slower in comparison to theaerobic degradation. Figure 5B shows the quantitative growthexperiments with depletion of DRO at a time course within 14days. The growth of strain MK2 was slow until 96 h of incubationand then reached a log phase by 100 h. The hydrocarbonoclasticpotential was closely coincided with the phase of cell growth, asa result, degradation efficiency increased from the 2nd day to the8th day of incubation, before leveling off from the 10th to the12th day. By the 10th day, a complete degradation of the substratehad occurred. Figure S2 depicts the residual DRO concentrationbefore and after incubation under anaerobic conditions.

Detection of Possible Catabolic Genes Involved in

Hydrocarbon DegradationThe presence of specific catabolic genes (alkB and the related,alkM, alkA) encoding alkane hydroxylase enzyme complexwas investigated by a PCR-mediated amplification with variousoligonucleotide primers. Of the 15 different oligonucleotidescombinations tested for PCR amplification, only the primercombination of the ALK3 set provided a positive result. Blastnsearches in the GenBank database showed that the PCR productwas similar to a number of known alkB genes, had a 96% matchwith the alkB gene encoding a putative alkane -1-monooxygenasefrom Burkholderia (Figure 6). In order to explore the presence ofother functional genes from the strain MK2, new primers weredesigned to amplify the second cluster of the alkane hydroxylasecomplex (Table S1). A PCR product of the expected size wasobtained when the primer combinations rubF, rubR used. Blastxalignments showed that this PCR product had 100% similarity tothe corresponding region of the S. maltophilia rubredoxin typeFe(Cys)4 protein (Figure 6).

DISCUSSION

The enrichment of hydrocarbonoclastic Electrode respiringbacteria (ERB) able to utilize hydrocarbons as a sole sourceof carbon and energy in MERS led to the isolation ofhydrocarbonoclastic bacterial strain identified as S. maltophiliaMK2. Stenotrophomonas spp. are often considered to beubiquitous, however, these species are frequently found inmarine, soil, rhizosphere of diverse plants (Denton and Kerr,1998) and polluted environments (Binks et al., 1995; Dunganet al., 2003; Lü et al., 2009) as their main environmentalreservoirs. The representative candidate, S. maltophiliaMK2 is a free living, facultatively anaerobic bacterium andphylogenetically placed in the phylum of Proteobacteria,Gammaproteobacteria, Xanthomonadales, Xanthomonodaceae(Palleroni and Bradbury, 1993). The environmental isolate

FIGURE 5 | (A) Biodegradation of DRO compounds by aerobically grown cells of S. maltophila MK2 (Blue circle shows DRO degradation in MK2 inoculated samples;

Red square shows the biomass density; Green triangle shows the DRO degradation in uninoculated controls). (B) Biodegradation of DRO compounds by

anaerobically grown cells of S. maltophila MK2 (Blue circle shows DRO degradation in MK2 inoculated samples; Red square shows the biomass density; Green

triangle shows the DRO degradation in uninoculated controls).




FIGURE 6 | Alignment of alkane monooxygenase (alkB) and rubredoxin (rubA) gene sequences generated using CLUSTALW multiple alignment in

S. maltophilia MK2.

S. maltophilia MK2 reduces nitrate in anoxic environmentsas reported earlier in some strains of this genus (Woodardet al., 1990). However, the additional distinctive features makethis strain different from the existing members of the familyinclude (i) growth by anaerobic fermentation of the aminoacids present in tryptone and peptone (ii) electrochemicallyactive under acetotrophic environments (iii) ability to degraden-alkane components of DRO in anaerobic conditions (iv)Biodecolorization of synthetic dyes. The regular growth mode ofthis bacterial strain S. malotophiliaMK2 is aerobic heterotrophy;however, the strain MK2 can grow in anaerobic environmentseither through amino acid fermentation or nitrate reduction. Theprevious studies on strain ZZ15 belongs to S. maltophilia showeda microaerophilic growth under denitrifying environments(Yu et al., 2009). In contrast, the pure cultures of manyStenotrophomonas strains are unable to grow in oxygen lackingconditions (Assih et al., 2002; Dungan et al., 2003).

Metabolic Versatility vs. EnvironmentalBioremediationBioremediation PotentialThe genus Stenotrophomonas has been studied as a promisingcandidate for biotechnological applications involved in thedetoxification of various man-made pollutants because of itsbroad spectrum of metabolic properties (Ryan et al., 2009). Theseinclude utilization of N-aromatic rings (Boonchan et al., 1998),alkyl benzene sulfonates of organophosphate pesticides (Dubeyand Fulekar, 2012), phenyl urea herbicides (Lü et al., 2009),chlorinated compounds (Somaraja et al., 2013), heavy metals(Pages et al., 2008; Ghosh and Saha, 2013) and other groups

of xenobiotic pollutants (Tachibana et al., 2003; Li et al., 2012).Aliphatic hydrocarbons including straight and cycloalkanes,unsaturated hydrocarbons and aromatic hydrocarbons, arethe building blocks of diesel oil (Air Force, 1989) and n-alkanes are the most dominant fraction. The degradation ofthese hydrocarbon compounds in anoxic environments bythe genus Stenotrophomonas is previously unknown. Here, wedemonstrate for the first time evidence for the occurrence ofhydrocarbonoclastic behavior in the strainMK2 under anaerobic,nitrate reducing environments.

The preliminary screening assays reveal that the strain MK2possess the hydrocarbonoclastic potential by involving redoxreactions in which electrons are donated to terminal electronacceptors during the cell respiration. The reduction of a lipophilicmediator such as DCPIP (blue to colorless) coupled with theformation of oxidized products showed that the biodegradationhad been carried out by metabolically active cell growth, not byadsorption to cells associated with the water-carbon interface(Kubota et al., 2008). The respiratory reduction of tetrazoliumsalts is another criterion employed by many researchers (Olgaet al., 2008; Pirôllo et al., 2008) to determine the dehydrogenaseactivity of hydrocarbonoclastic bacterial strains. Upon reductionof this salt, the color changed to red due to the formation ofinsoluble formazans by the production of superoxide radicalsand electron transport in the bacterial respiratory chain (Haineset al., 1996). In order to corroborate the potential hydrocarbondegradation by the strain MK2, GC scan was performed usingheterotrophically incubated samples grown under aerobic andanaerobic conditions. The highest rate of degradation of thelight end hydrocarbons of DRO was observed at 24 h withaerobic incubations, whereas this tended to be slower (96 h)




under anaerobic conditions. GC resolved n-alkanes from C8to C36 peaks (Figure S1) in inoculated samples demonstratedthe occurrence of the enhanced hydrocarbon degradation whenthe bacterial strain grown under the aerobic conditions. It wasquite possible to achieve a complete degradation of DRO underaerobic conditions by appropriately increasing the incubationtime of the experiment. Such hydrocarbonoclastic behavior isin contrast to the earlier findings of Saadoun (2002) and Uenoet al. (2007) where their strain of S. maltophilia was unable todegrade hydrocarbons as a sole carbon source. On the otherhand, members of this genus have been found along withother predominant genera of hydrocarbon degraders includingAcinetobacter, Pseudomonas, Alcaligenes, Sphingomonas in oilcontaminated environments as stated earlier (Van Hamme et al.,2000; Zanaroli et al., 2010). The previous studies on the microbialelectrochemical remedial process of hydrocarbons have alsodemonstrated the ubiquity of Stenotrophomonas spp. and theirdominance in the anodic microbial communities (Morris et al.,2009; Venkidusamy et al., 2016). The capability of hydrocarbondegradation has also been demonstrated earlier in a soil isolate ofS. maltophilia strain DJLB only under aerobic conditions (Ganeshand Lin, 2009). It is of interest that, the present study reveals thecomplete mineralization of n-alkane members of DRO (C8–C36)for the first time, including the biomarkers pristine, phytane,and a short chain to long chain aliphatic hydrocarbons underanaerobic incubations by the strain MK2 during a 12 days periodin the presence of nitrate.

Exoelectrogenic PotentialThe characteristics of diazoic dye decolorization were used as asimple criterion for pre-screening the possible electrochemicallyactive microbial candidates in the present study as stated earlier(Hou et al., 2009). The present study showed the simultaneousdecolorization and decreased dye concentrations from batchculture studies of anaerobic and aerobic incubations with strainMK2 inoculum. The efficiency of color removal was morethan 95% under anaerobic conditions as reported in anotherexoelectrogenic strain of Shewanella spp. (Pearce et al., 2006).This is in agreement with the previous studies on the assessmentof electrochemically active microbial strains using MFC arrays(Hou et al., 2009). Dye decolorization occurs because of areductive electrophilic cleavage of the chromophore, a functionalgroup of azo linkage, by biocatalysts as reported earlier (Sunet al., 2009; Satapanajaru et al., 2011). To confirm the extracellularaccess to the insoluble electron acceptors, the exoelectrogenicproperty of the strain MK2 was also investigated in twodifferent environments (i) acetotrophic (ii) dye decolorization,using microbial electrochemical systems. The present studyexhibited a maximum power density of 113 ± 7 mW/m2 witha recovery of 34.8% as an electrical current using the strainMK2 in acetotrophic conditions. In the case of the reactorsfed with azoic dye demonstrated the potential of generatingcurrent (99.93± 6 mW/m2) with the concurrent decolourizationusing the strain MK2 in MFCs for the first time. The resultspresented in this study suggest that the strain MK2 is capable ofutilizing insoluble electron acceptors such as electrodes throughextracellular electron transfer mechanisms. Recent investigations

have revealed the potential of using such pure cultures ofheterotrophic biofilms in microbial electrochemical remediationcells for dye detoxification (Chen et al., 2010a,b). For instance,Chen et al. (2010a), reported the possibility of using pure culturesof Proteus hauseri inMFC, however, decolorization efficiency andpower densities generated were much lower. The performanceof these microbial electrochemical cells using pure cultures ofexoelectrogens are considerably affected by a number of reactorparameters and operating conditions as reported earlier (Minet al., 2005; Logan, 2008).

Genetic PotentialTo gain deeper insights into the hydrocarbon degradationmechanism by the strainMK2, we carried out a gene specific PCRanalysis to identify the possible catabolic genes encoding alkanedegrading enzymes using different degenerate oligonucleotides.The mechanisms of these alkanes activation vary accordingto the lifestyle of representative candidate microorganismsand growth environments. Under aerobic environments, thebiodegradation typically occurs through a sequential oxidationof n-alkanes resulting in corresponding alcohol and aldehydesgroups. These aldehydes further metabolized into fatty acidsand conjugated with CoA through β oxidation process whichthen enter into the tricarboxylic acid cycle as shown in Figure 7(Van Hamme et al., 2003; van Beilen et al., 2004). Such asuccessional oxidation process is activated by a family of integralmembrane proteins called alkane hydroxylase enzyme system,and this was first studied in Pseudomonas putida GPo1. Thisis of particular interest being a three component biocatalystand composed of alkane monooxygenase (alkB group), dinucleariron rubredoxins (rubA, rubB) and mononuclear rubredoxinreductase (rubR) (Rojo, 2009; Teimoori et al., 2011). Thesegenes encode the alkane hydroxylase (alk) system in the enzymecomplex which activates the terminal carbon atoms in the chainof hydrocarbons.While searching the catabolic genes that encodealk system in the strain S. maltophilia MK2, we found forthe first time a conserved chromosomal region of alkB andrubA (Figure 6). Insilco analysis of this gene showed that thealkB region was highly similar to the region observed froman alkB gene of Burkholderia spp. and this is presumably thegene providing this activity, supporting the close relationshipbetween S. maltophilia and Burkholderia spp. (84%) at thegenomic level. This result suggests that the genes involved do notcorrespond in terms of their sequence to the same genes as perthe published Stenotrophomonas malotophilia genome and wereinstead derived from some other organism with different genesequences (and the discovery that the alkB gene sequence comesfrom Burkholderia supports this). In contrast, the earlier studieson catabolic genes for alkane degradation in Stenotrophomonasspp. have shown negative results for the amplification of alkBgene (Smits et al., 1999; Vomberg and Klinner, 2000). Thepresence of a rubA gene with 100% homology to Rubredoxin-type Fe(Cys)4 protein of S. maltophilia R551-3, shows that thebacterial strainMK2 likely possesses an essential electron transfercomponents for alkane hydroxylation. Together, these resultsperhaps indicate the presence of the two conserved domains ofalkB-rubA fused proteins in a contiguous open reading frame as




FIGURE 7 | Degradation pathways of DRO compounds in aerobic environments.

shown earlier in metagenomic analysis of alk genes of differentmicrobial genomes from diverse environments (Nie et al., 2014).Such a fusion would be responsible for the extended spectrum ofalkane degradation up to C36 hydrocarbons shown in the presentstudy, as alkB often reported to be responsible for


the phylogeny knowledge on bioleaching agents and showingfurther potential in the treatment of wastewater from textileindustries using MERS. On a global scale, the strain providesmany exciting opportunities for increasing our understandingon bioremediation that underpins the molecular mechanismof contaminant degradation in a relevant environmentalcontext.

AUTHOR CONTRIBUTIONS

KV and MM proposed the study. KV conducted the experimentsunder the supervision of MM. KV prepared the draft withcontributions from MM.

ACKNOWLEDGMENTS

The authors thank Dr. R. Lockington for comments andsuggestions on previous versions of this manuscript. KV thanksAustralian Federal Government, University of South Australiafor International Postgraduate scholarship award (IPRS) andCRC CARE for the research top-up award.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be foundonline at: http://journal.frontiersin.org/article/10.3389/fmicb.2016.01965/full#supplementary-material

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Identification of Electrode Respiring, Hydrocarbonoclastic Bacterial Strain Stenotrophomonas maltophilia MK2 Highlights the Untapped Potential for Environmental BioremediationIntroductionMaterials and MethodsSource of ChemicalsBacterial Strain, Media, and Culture ConditionsBacterial 16S rRNA Gene SequencingAssessment of Biodegradation Potential and Electrochemical ActivityHydrocarbon Biodegradation ExperimentsFuel Cell ExperimentsMFC Construction and Operational Conditions

Cloning and Phylogenetic Analysis of Possible Catabolic Genes for Hydrocarbon DegradationAnalytical Methods and Calculations

ResultsStrain Isolation and PhysiologyPhylogenetic Analysis and TaxonomyRedox Indicator Assays for the Assessment of Hydrocarbonoclastic PotentialScreening Assays for the Assessment of Electrochemical ActivityEnergy Generation by S. maltophilia MK2 in Microbial Electrochemical CellsCurrent Generation in Acetate Fed MFCsCurrent Generation and Simultaneous Dye Decolorization in Dye Fed Microbial Electrochemical Cells

Hydrocarbonoclastic Potential of S. maltophilia MK2Aerobic Biodegradation of DRO

Anaerobic Biodegradation of DRODetection of Possible Catabolic Genes Involved in Hydrocarbon Degradation

DiscussionMetabolic Versatility vs. Environmental BioremediationBioremediation Potential

Exoelectrogenic PotentialGenetic Potential

ConclusionsAuthor ContributionsAcknowledgmentsSupplementary MaterialReferences

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